CN112611876A - Aptamer-quadruplex urine amino acid detection kit and method - Google Patents
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Abstract
The invention belongs to the field of medical detection, and particularly relates to a nucleic acid aptamer-quadruplex urine amino acid detection kit and a method. The invention discloses a nucleic acid aptamer-quadruplex urine amino acid detection kit, which comprises a certain amino acid specific nucleic acid aptamer and a DNA G quadruplex sequence, and is characterized in that the certain amino acid specific nucleic acid aptamer is complementary with a partial sequence of the DNA G quadruplex sequence, the certain amino acid specific nucleic acid aptamer and the DNA G quadruplex sequence form a complementary binding sequence under the condition that no target amino acid exists, and the DNA G quadruplex sequence cannot form a G quadruplex. The method is simple and convenient to operate, has high analysis speed, can detect the concentration of the amino acid in the nm-mum level range in a reaction system, and is favorable for clinical development and screening of high-incidence areas of gastric cancer.
Description
Technical Field
The invention belongs to the field of medical detection, and particularly relates to a nucleic acid aptamer-quadruplex urine amino acid detection kit and a method.
Background
Metabonomics is a newly developed subject after genomics and proteomics, and researches the occurrence and development of diseases, searches disease biomarkers, predicts disease prognosis and the like by accurately, qualitatively and quantitatively determining small molecular metabolites in human bodies and analyzing the relation between the metabolites and physiological and pathological changes of the human bodies. Metabonomics has wide development prospect in clinical diagnosis, and has four main application directions: the application in clinical diagnosis, research application in etiology and pathological mechanism, application in clinical medication guidance, and application in preclinical animal model screening.
The Chinese invention patent (CN201310556941.9) discloses that a group of raised small molecular amino acids can be used as a marker for screening gastric cancer in urine of a gastric cancer patient by a gas chromatography-mass spectrometry combined technology. The new discovery of urine small molecule biomarkers makes noninvasive gastric cancer screening and diagnosis possible. However, amino acids found in urine of gastric cancer patients at the early stage belong to small molecular substances, and high-grade precise instruments such as a gas chromatography/mass spectrometry (GC/MS) analyzer are required, and the instruments are not clinically conventional instruments, so that the instrument is difficult to popularize in clinic and is more difficult to apply to population general investigation of high-incidence areas of gastric cancer.
Disclosure of Invention
The invention aims to provide a nucleic acid aptamer-quadruplex urine amino acid detection kit and a method, so as to meet the requirement of non-invasive gastric cancer screening.
The invention discloses a nucleic acid aptamer-quadruplex urine amino acid detection kit, which comprises a certain amino acid specific nucleic acid aptamer and a DNA G quadruplex sequence, and is characterized in that the certain amino acid specific nucleic acid aptamer is complementary with a partial sequence of the DNA G quadruplex sequence, the certain amino acid specific nucleic acid aptamer and the DNA G quadruplex sequence form a complementary binding chain under the condition that no target amino acid exists, and the DNA G quadruplex sequence cannot form a G quadruplex.
The technical principle of the invention is that based on the fact that the binding capacity between the aptamer and the target amino acid is larger than the interaction force between the aptamer and the complementary DNA G quadruplex sequence, the aptamer sequence targeting a certain amino acid is hybridized with the DNA G quadruplex sequence; then adding amino acid into the reaction system; at this time, the aptamer sequence specifically binds to the target amino acid, and the bound DNA G quadruplex sequence is released; and adding heme molecules into the system, wherein the heme and the DNA G quadruplex sequence form a DNAzyme with catalytic activity, and the HRP activity of the DNAzyme can be used for catalyzing a mixed solution of hypersensitive chemiluminescent liquid A, B to react to generate a chemiluminescent signal or catalyzing DAB to generate a brown color substance, so that the formed chemiluminescent light density or color intensity is in direct proportion to the content of target amino acid molecules (figure 1).
In a preferred embodiment, the amino acid is serine.
In a preferred embodiment, the nucleic acid sequence of the aptamer is shown in Seq ID No. 1.
In a preferred embodiment, the DNA G quadruplex sequence has the nucleic acid sequence shown as Seq ID No. 2.
In a preferred embodiment, the urine amino acid detection kit further comprises a hypersensitive chemiluminescent liquid or a DAB color-developing liquid;
on the other hand, the invention also discloses a DNA G quadruplex sequence which is complementary and matched with the serine-specific aptamer sequence, and the nucleic acid sequence is shown in Seq ID No. 2;
on the other hand, the invention also discloses the application of the DNA G quadruplex sequence in preparing a gastric cancer diagnostic reagent.
On the other hand, the invention also discloses a method for detecting single amino acid in urine, which comprises the following steps:
an amino acid aptamer hybridizes to the DNA G quadruplex sequence;
adding a urine sample to be detected;
adding a heme reagent;
adding hypersensitive chemiluminescent liquid or DAB color developing liquid;
detecting chemiluminescence or colorimetry.
In a preferred embodiment, the amino acid is serine.
In a preferred embodiment, the nucleic acid sequence of the aptamer is shown in Seq ID No. 1.
In a preferred embodiment, the DNA G quadruplex sequence has the nucleic acid sequence shown as Seq ID No. 2.
The method is simple and convenient to operate, has high analysis speed, can detect the concentration of the amino acid in the nm-mum level range in a reaction system, and is favorable for clinical development and screening of high-incidence areas of gastric cancer.
Drawings
FIG. 1 is a schematic diagram of the basic principle of the present invention;
FIG. 2 is a regression curve of the results of detection of serine standards;
FIG. 3 is the results of clinical samples tested using the method of the invention;
FIG. 4 is a chromatogram showing the results of amino acid analysis of a urine sample from a patient with gastric cancer (positive case by the detection method of the present invention);
FIG. 5 is a chromatogram showing the results of detection of a urine sample from a physical examiner by an amino acid analyzer (negative cases are shown by the detection method of the present invention).
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
1. Instruments and reagents
QT-1 vortex instrument (Shanghai Qite analytical instruments Co., Ltd.)
Water purification device (MILLI-XQ, Murrill Biotechnology Co., Ltd., China)
Mini shaking bed 2211k (Taicang city science and education equipment factory)
Full-wavelength multifunctional microplate reader Safire 2(Tecan, Switzerland)
Aptamer sequence (Shanghai Biometrics, Ltd., purified by PAGE)
Taking the serine aptamer as an example (Seq ID No. 1: 5'-TGGGACAACATCAGACAGGTCGTTTGATGTCAGATTGGCCAGGTC-3', Shanghai Biometrics Ltd., PAGE purification)
DNA G quadruplex oligonucleotide sequence (Seq ID No. 2: 5'-CCC AGG TTT GGG TAG GGC GGG TTG GG-3', Shanghai Biometrics Ltd., HPLC purification)
Heme (Hemin, 51280-1G, sigma Sigma, Germany)
Bovine serum albumin BSA; tween 20, NaCl, MgCl2, Tris
Dimethyl sulfoxide DMSO (A503039, Shanghai Sheng Gong)
Hypersensitive chemiluminescent A, B liquid (MA0186-L, Meilun biology)
Amino acids (Purity > 98.5%, LR, BBI Life Sciences, China)
DAB reagent (Meilun biology, 3ml, MA0201)
2. Preparation of main reagent
2.1DNA molecular hybridization solution (Tris-HCl buffer)
2.42g Tris (LOT #: O1008A, Melongena, China), 17.55g NaCl (MB2471, Melongena, China) and 1.00g MgCl 2.6H 20(LOT: M0304A, Melongena) were dissolved in 500ml ultrapure water, the pH was adjusted to 8.3 with 0.1M HCl, and finally diluted with ultrapure water to 1000ml Tris-HCl solution for storage at room temperature.
2.2 Hemin stock solution of heme
Hemin heme is dissolved in DMSO, dissolved at 10 μ M and stored at 4 deg.C.
2.3 hypersensitive chemiluminescent A, B liquid
The solution A and the solution B are prepared according to the proportion of 1: 1.
2.4 amino-terminal gradient solutions
Amino acids (here, serine is taken as an example) are dissolved sufficiently in a certain amount of sterilized deionized ultrapure water, and then prepared into a certain concentration gradient (0, 2.5, 5, 10, 20, 40, 80, 160. mu.g/ml), and stored at 4 ℃.
2.5 amino acid aptamers
Here, taking serine aptamer as an example, the aptamer for amino acid was dissolved in sterilized deionized ultrapure water to form a 50. mu.M solution and used as it is.
3. Sample to be tested
64 morning midstream urine samples of the subjects (36 gastric cancer hospitalized patients, 28 contemporaneous health examiners) were collected. The urine sample was placed in a 15ml retention tube and centrifuged at 3000RPM for 5 minutes with the lid closed, and the supernatant was pipetted into a 1.5ml EP tube for dispensing and stored in a deep freezer at-80 ℃ until use.
Example 1 urinary serine assay
The serine-targeting aptamer solution was mixed well with the G quadruplex DNA in Tris-HCl buffer at a quantitative ratio of 5: 4. In the detection system, a concentration gradient standard substance is firstly set according to the possible concentration range of the amino acid to be detected in a sample. Taking serine as an example, 8 serine concentration gradient standards are 0, 2.5, 5, 10, 20, 40, 80 and 160 mu g/ml. mu.L (concentration 50. mu.M) of the aptamer and 96. mu.L of the G quadruplex DNA (concentration 50. mu.M) were hybridized in 600. mu.L of Tris-HCl solution (pH 8.3), and reacted on a vortex apparatus in a 37 ℃ water bath for 1 hour for complete reaction. After the reaction is finished, 75 mu l of hybridization mixed solution is added into each 1.5ml centrifuge tube, 75 mu l of serine standard substance or sample to be detected with each concentration gradient is added, and the mixture is placed in a water bath at 37 ℃ to be fully mixed and reacted for 1 h. Then, 16. mu.l of a hemoglobin solution (stock concentration: 10. mu.M) was added to the tube, and the mixture was mixed well until the final concentration was 1. mu.M, and reacted in a water bath at 37 ℃ for 3 hours in the absence of light. Then 20 mul of prepared luminescent liquid AB mixed solution is added into each tube and fully mixed to be used as a luminescent catalytic substrate. Finally, each tube of the above liquid was added to three sub-wells of a 96-well plate in a volume of 50 μ L per well. And (3) placing the 96-well plate in a Safire 2 full-wavelength multifunctional microplate reader for detection.
And (3) dividing the detection mean value of the three auxiliary holes of the sample to be detected with the blank control mean value of the standard curve for normalization treatment, and carrying out difference comparison among different groups by using unpaired t-test.
In order to compare and analyze the sensitivity of the urine amino acid detection system, the invention simultaneously adopts a standard amino acid analysis instrument to carry out parallel detection. The main detection process is as follows: mu.l urine and 500. mu.l pretreatment solution (0.1M hydrochloric acid and 10% trichloroacetic acid) are added into a 1.5ml EP tube according to the proportion of 1:2, fully and uniformly mixed, centrifuged at 12000RPM and 4 ℃ for 20 minutes, then kept stand at 4 ℃ for 30 minutes, and centrifuged at 12000RPM and 4 ℃ for 20 minutes. Taking 1ml of the centrifuged supernatant, adding the supernatant into a sample bottle of a full-automatic free amino acid analyzer for machine detection (Hitachi, Japan) and taking the supernatant as a reference for a subsequent detection result.
The experimental results are as follows:
1. standard curve
Chemiluminescence values measured from 8 standard concentration gradients (0, 2.5, 5, 10, 20, 40, 80, 160 μ g/ml) as internal controls showed a substantially linear relationship with increasing serine concentration (fig. 2).
2. Clinical sample test results
A total of 64 urine samples were tested (28 of them were healthy examiners, and 36 were gastric cancer patients). In order to eliminate the difference between detection batches, the actual chemiluminescence detection value is divided by the mean value of the luminescence values of the standard substances in the same batch for normalization. The detection result is shown in fig. 3, the serine detection value of the urine sample of the gastric cancer patient is significantly different from that of the healthy examinee, and represents that P is less than 0.01.
3. Evaluation of the method
To evaluate the detection method of the present invention, we performed a parallel analysis of the same batch of samples using an amino acid detector, with clinical diagnosis as "gold standard" as a reference (all gastric cancer patients were confirmed by pathological diagnosis after surgery).
Detection method the cut-off value of the detection result (cut-off) was obtained with a 95% confidence interval. Wherein the method of the invention has a cutoff value of 4.28, above which a positive is defined and below which a negative is defined. The amino acid detector method detects a cutoff value of 50.94 above which a positive is defined and below which a negative is defined. Typical case results of the amino acid detector analysis used as a reference are shown in FIG. 4 and FIG. 5.
A four-table (table 1) between the diagnosis of gastric cancer and the gold standard by the detection method of the invention is calculated, which shows that the sensitivity of the method of the invention is 77.78%, the specificity is 85.71% and the accuracy is 81.25%; the detection result of the amino acid detector as a reference and a clinical diagnosis four-table (table 2) have the sensitivity of 25 percent, the specificity of 82.14 percent and the accuracy of 50 percent; .
TABLE 1 four-grid table of the detection method of the present invention
TABLE 2 four-grid table of amino acid analyzer detection method
The detection result of taking serine as a target molecule shows that the urine amino acid detection system established by the invention has higher sensitivity and is more beneficial to clinical development and screening of high-incidence areas of gastric cancer.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Sequence listing
<110> Renjin Hospital affiliated to Shanghai university of transportation medical school
<120> aptamer-quadruplex urine amino acid detection kit and method
<141> 2020-12-30
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tgggacaaca tcagacaggt cgtttgatgt cagattggcc aggtc 45
<210> 2
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cccaggtttg ggtagggcgg gttggg 26
Claims (10)
1. A nucleic acid aptamer-quadruplex urine amino acid detection kit comprises a certain amino acid specific nucleic acid aptamer and a DNA G quadruplex sequence, and is characterized in that the certain amino acid specific nucleic acid aptamer is complementary with a partial sequence of the DNA G quadruplex sequence, the certain amino acid specific nucleic acid aptamer and the DNA G quadruplex sequence form a complementary binding strand in the absence of target amino acids, and the DNA G quadruplex sequence cannot form a G quadruplex.
2. The urine amino acid detection kit of claim 1, wherein the amino acid is serine.
3. The urine amino acid detection kit according to claim 1, wherein the nucleic acid sequence of the aptamer is Seq ID No. 1.
4. The urine amino acid detection kit of claim 1, wherein the nucleic acid sequence of the DNA G quadruplex sequence is Seq ID No. 2.
5. The urine amino acid detection kit according to claim 1, further comprising a super-sensitive chemiluminescent liquid or a DAB color-developing liquid.
6. A DNA G quadruplex sequence which is partially complementary paired with a serine specific aptamer sequence, characterized in that its nucleic acid sequence is Seq ID No. 2.
7. Use of the DNA G quadruplex sequence according to claim 6 for the preparation of a diagnostic agent for gastric cancer.
8. A method for detecting single amino acid in urine is characterized by comprising the following steps:
(1) an amino acid aptamer hybridizes to the DNA G quadruplex sequence;
(2) adding a urine sample to be detected;
(3) adding a heme reagent;
(4) adding hypersensitive chemiluminescent liquid or DAB color developing liquid;
(5) detecting chemiluminescence or colorimetry.
9. The detection method according to claim 8, wherein the certain amino acid is serine.
10. The detection method according to claim 8, wherein the nucleic acid sequence of the aptamer is represented by Seq ID No.1 and the nucleic acid sequence of the DNA G quadruplex sequence is represented by Seq ID No. 2.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105821045A (en) * | 2016-05-24 | 2016-08-03 | 上海敬元投资有限公司 | Nucleic-acid aptamer for L-serine and application thereof |
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| CN105821046A (en) * | 2016-05-24 | 2016-08-03 | 上海敬元投资有限公司 | Specific L-serine aptamer and application thereof |
| WO2016168656A1 (en) * | 2015-04-15 | 2016-10-20 | Ji Hoon Lee | Aptasensor and method of detecting target material |
| CN110186892A (en) * | 2019-06-26 | 2019-08-30 | 苏州健雄职业技术学院 | A kind of glutamine detection method based on dual-enzyme coupling |
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| WO2016168656A1 (en) * | 2015-04-15 | 2016-10-20 | Ji Hoon Lee | Aptasensor and method of detecting target material |
| CN105821046A (en) * | 2016-05-24 | 2016-08-03 | 上海敬元投资有限公司 | Specific L-serine aptamer and application thereof |
| US20190292580A1 (en) * | 2018-03-23 | 2019-09-26 | Rutgers, The State University Of New Jersey | Dna logic-gated proximity assembly circuit for biochemical sensing |
| CN110186892A (en) * | 2019-06-26 | 2019-08-30 | 苏州健雄职业技术学院 | A kind of glutamine detection method based on dual-enzyme coupling |
| CN111500684A (en) * | 2020-05-14 | 2020-08-07 | 河北医科大学 | Method for detecting dopamine based on aptamer-G quadruplex nanowire |
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| 修风民 等: "胃癌患者血浆 24 种氨基酸代谢物水平变化", 《第二军医大学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105821045A (en) * | 2016-05-24 | 2016-08-03 | 上海敬元投资有限公司 | Nucleic-acid aptamer for L-serine and application thereof |
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